Apparatus and method for preparing cosmeceutical ingredients containing epi-dermal delivery mechanisms

ABSTRACT

The skin serves as a barrier that protects the body from the external environment and prevents water loss. This barrier function also prevents most hydrophilic or hydrophobic and large molecular weight ingredients (&gt;500 kDa) from penetrating intact skin. Until recently, methods to increase stratum corneum permeability were generally not effective enough to make the stratum corneum so permeable that the barrier posed by the viable epidermis mattered. However, that has now changed with the development of the present embodiment&#39;s physical methods and highly optimized chemical formulations, such that we revisited the permeability of the full epidermis with the example embodiment&#39;s constructs and not focus only on the stratum corneum. This example embodiment therefore tests the hypothesis that the viable epidermis offers a significant permeability barrier to both small molecules and macromolecules that becomes the rate limiting step.

PRIORITY APPLICATION

This is a continuation patent application drawing priority from U.S.patent application Ser. No. 14/971,320; filed Dec. 16, 2015. Thispresent patent application draws priority from the referenced patentapplication. The entire disclosure of the referenced patent applicationis considered part of the disclosure of the present application and ishereby incorporated by reference herein in its entirety.

TECHNICAL FIELD

The application of innovative micro and nano vesicle formingtechnologies to effect beneficial results through the application ofsynthetic and natural ingredients to the skin has shown a greatpotential to significantly benefit the cosmetic formulation practice,offering solutions to many of the current limitations in ingredients,treatment style and management of human skin effected by environmentaland physiological impact.

BACKGROUND

A liposome vesicle encapsulates a region of aqueous solution inside ahydrophobic membrane; dissolved hydrophilic solutes cannot readily passthrough the lipids. Hydrophobic chemicals can be dissolved into themembrane, and in this way liposome can carry both hydrophobic moleculesand hydrophilic molecules.

Several CFs (Compressed Fluid) methodologies have been used to generatevesicles, some of them already existed and others were developed forthis specific application. Most of the methods involve a mixture betweenthe compressed CO2, the vesicle membrane constituents and an organicsolvent for producing the vesicles upon contact with an aqueous phase.

Depending on the role of the compressed CO2 used in each method, theycan be classified as: Process involving the use of CO2 as a solvent(e.g. Supercritical Liposome Method and Rapid Expansion of SupercriticalSolutions), Processes involving the use of CO2 as an antisolvent (e.g.Gas Antisolvent Precipitation and Aerosol Solvent Extraction System) andProcesses involving the use of CO2 as a co-solvent or a processing aid(e.g. Depressurization of an Expanded Liquid Organic Solution-Suspensionand Supercritical Reverse Phase Evaporation).

Model hydrophilic and hydrophobic compounds, such as fluorescent dyes,sugars and cholesterol, have been encapsulated into vesicles using thesemethodologies whereas biomolecules like proteins, anticancer drugs andantibiotic, have been integrated in less extent.

Transdermal delivery systems (TDS) were introduced onto the US market inthe late 1970s), but transdermal delivery of drugs had been around for avery long time. There have been previous reports about the use ofmustard plasters to alleviate chest congestion and belladonna plastersused as analgesics. The mustard plasters were homemade as well asavailable commercially where mustard seeds were ground and mixed withwater to form a paste, which was in turn used to form a dispersion typeof delivery system.

Once applied to the skin, enzymes activated by body heat led to theformation of an active ingredient (allyl isothiocyanate). Transport ofthe active drug component took place by passive diffusion across theskin—the very basis of transdermal drug delivery.

The epi-dermis undergoes changes in structure and function which resultin many of the characteristics of aged skin, including loss ofelasticity, formation of wrinkles, loss of water-holding capacity,sagging, and poor microcirculation. At the molecular level, thesechanges have been correlated with biochemical changes in the content andstructure of the extracellular matrix to which the major cells of theepi-dermis (i.e., the fibroblasts) reside. Collagen becomes highlycross-linked and inelastic, elastin is reduced in amounts and isincorrectly distributed, which results in reduced intercellular waterfor reduction and repair of these changes. Nonsurgical options includechemical peels and chemicals with minor irritant properties (e.g.,topical retinoid, salicylic acid, and alpha-hydroxy acids), are based onthe principle of wounding the stratum corneum—the skin's primary defenseagainst the transit of exogenous materials into the epidermis anddermis—to allow the penetration of constituents through the disruptedskin, which stimulates the desired response, typically restorativehealing. All of these techniques require a wound healing response to theskins being intentionally wounded as a method to initiate therejuvenation process.

Owing to the selective nature of the skin barrier, only a small pool ofingredients can be delivered non-systemically or systemically attherapeutically relevant rates. Besides great potency, thephysicochemical ingredient characteristics often evoked as favorable forpercutaneous delivery include moderate lipophilicity andlow-molecular-weight. However, a large number of skin damage mitigatingactive agents do not fulfill these criteria.

Chemical permeation enhancers facilitate drug permeation across the skinby increasing drug partitioning into the barrier domain of the stratumcorneum, increasing drug diffusivity in the barrier domain of thestratum corneum or the combination of both (2).

The heterogeneous stratum corneum is composed of keratin ‘bricks’ andintercellular continuous lipid ‘mortar’ organized in multilamellarstrata (3)(4)(5). Depending on the nature of the drug or ingredient,either of these two environments may be the rate-limiting milieu(barrier domain) for the percutaneous transport.

As a consequence, it is anticipated that the magnitude of permeationimprovement obtained with a given permeation enhancer will vary betweenlipophilic and hydrophilic ingredients. Several mechanisms of action areknown: increasing fluidity of stratum corneum lipid bilayers, extractionof intercellular lipids, increase of ingredient's thermodynamicactivity, increase in stratum corneum hydration, alteration ofproteinaceous corneocyte components and others.

The stratum corneum is a formidable barrier to exogenous agentsincluding cosmeceutical ingredients. Therefore, it is often necessary toadd permeation-enhancing chemicals to aid beneficial constituents inpassing through the stratum corneum. Permeation-enhancing chemicalsinclude fatty acids, organic solvents (i.e., acetone and ethanol),alcohols, esters and surfactants.

It is generally understood that for enhancers, increased potency isdirectly correlated with increased skin irritation. Difficulty inreducing the irritation of these agents has been expressed since thesame mechanisms responsible for increasing permeation cause irritation.While potent enhancers are effective at transiently compromising theintegrity of the stratum corneum barrier, their action is not entirelylimited to the stratum corneum and the interaction with viable epidermiscan cause cytotoxicity and irritation. Published methods for reducingthe skin irritation of permeation enhancers include combining permeationenhancers (synergistic mixtures) and manipulation of their chemicalstructures.

Conventional lipid or niosome vesicle production techniques havedrawbacks such as complex and time consuming procedures involvingorganic solvents. For liposomes, conventional methods can involve harshconditions that result in denaturation of the lipids and activeingredients, and also cause poor ingredient encapsulation efficiency.

Since the liposomes were first used as drug carriers in 1970s. Manymethods, such as Supercritical fluids (SCFs), for preparing liposomeshave been developed, but these methods require large amounts of organicsolvents like chloroform, ether, freon, methylenechloride and methanolthat are harmful to the environment and the human body, and very fewmethods have been developed that yield liposomes that have a hightrapping efficiency for water soluble substances without using anyorganic solvent.

Additionally, all these methods are not suitable for mass production ofliposomes because they consist of many steps. With the advent of GreenChemistry in the early 1990s, the surge of supercritical fluids (SCFs)increased vastly.

The supercritical state of a fluid (SCF) is intermediate between that ofgas and liquids. The SCF has been used widely in pharmaceuticalindustrial operations including crystallization, particle sizereduction, drug delivery preparation, coating and product sterilization.In the pharmaceutical field, supercritical carbon dioxide (scCO2) is byfar the most commonly used gas, which can become supercritical atconditions that are equal or exceed its critical temperature of 31.1° C.and its critical pressure of 7.38 Megapascals (Mpa).

The encapsulation degree of any drug into vesicles is influenced byseveral parameters related to the: a) vesicle composition, b) the natureof the cosmeceutical ingredient and c) the preparation methodology.Regarding the vesicle composition, besides the selection of the lipidsforming the membrane and the presence of charges on it, the type ofvesicle plays also an important role. Thus, for hydrophilic drugs, suchas proteins or peptides, the encapsulation degree appears to increase inthe following order: MLV<SUV<LUV. (FIG. 1.0) Nevertheless in the case ofhydrophobic drugs, the size and type of liposomes do not seem to play amajor role.

Liposomes with a single bilayer are known as unilamellar vesicles (UV).UVs may be made extremely small (SUVs) or large (LUVs) (FIG. 3.0).Liposomes are prepared in the laboratory by sonication, detergentdialysis, ethanol injection, French press extrusion, ether infusion, andreverse phase evaporation.

These methods often leave residuals such as detergents or organics withthe final liposome. From a production standpoint, it is clearlypreferable to utilize procedures which do not use organic solvents sincethese materials must be subsequently removed.

Some of the methods impose harsh or extreme conditions which can resultin the denaturation of the phospholipid raw material and encapsulatedingredients. These methods are not readily scalable for mass productionof large volumes of liposomes.

Several methods, such as energy input in the form of sonic energy(sonication) or mechanical energy (extrusion), exist for producing MLVs(multilamellar vesicles), LUVs and SUVs without the use of organicsolvents.

MLVs (multilamellar vesicles), free of organic solvents, are usuallyprepared by agitating lipids in the presence of water. The MLVs are thensubjected to several cycles of freeze thawing in order to increase thetrapping efficiencies for water soluble ingredients.

MLVs are also used as the starting materials for LUV and SUV production.One approach of creating LUVs, free of organic solvents, involves thehigh pressure extrusion of MLVs through polycarbonate filters ofcontrolled pore size. SUVs can be produced from MLVs by sonication,

French press or high pressure homogenization techniques. High pressurehomogenization has certain limitations. High pressure homogenization isuseful only for the formation of SUVs. In addition, high pressurehomogenization may create excessively high temperatures.

Contrary to the present embodiment, extremely high pressures areassociated with equipment failures. High pressure homogenization doesnot insure end product sterility. High pressure homogenization isassociated with poor operability because of valve plugging and poorsolution recycling.

The use of liposomes for the delivery and controlled release oftherapeutic drugs requires relatively large supplies of liposomessuitable for in vivo use (FIG. 6.0). Present laboratory scale methodslack reproducibility, in terms of quantity and quality of encapsulatedingredients, lipid content and integrity, and liposome size distributionand captured volume.

The multidimensional characteristics of the ingredient and the liposome,as well as potential raw material variability, influencereproducibility. Present state-of-the-art liposome and niosome productsare not stable. It is desirable to have final formulations which arestable for six months to two years at room temperature or atrefrigeration temperature.

Present liposome products are difficult to sterilize. Sterility iscurrently accomplished by independently sterilizing the component partslipid, buffer, ingredient and watery autoclave or filtration and thenmixing in a sterile environment.

This sterilization process is difficult, time consuming and expensivesince the product must be demonstratively sterile after severalprocessing steps. Heat sterilization of the finished product is notpossible since heating liposomes or niosomes does irreparable damage.Filtration through 0.22 micron filters may also alter the features ofmultilayered liposomes and elastic niosomes.

Gamma ray treatment, not commonly used in the pharmaceutical industry,may disrupt liposome or elastic niosome membranes. Picosecond lasersterilization is still experimental and has not yet been applied to thesterilization of any commercial pharmaceutical.

In the past two decades, several cosmetic formulations based oningredient delivery systems have been successfully introduced for thetreatment of skin disorders. Many problems exhibited by free activecosmetic ingredients (ACIs), such as poor solubility, toxicity, rapid invivo breakdown, unfavorable pharmacokinetics, poor bio distribution andlack of selectivity for target tissues can be ameliorated by the use ofa VDS (vesicle delivery system) as offered by the current embodiment.Although a whole range of delivery agents exist nowadays, the maincomponents typically include a nanocarrier, a targeting moietyconjugated to the nanocarrier, and a cargo, such as the desiredcosmeceutical ingredient.

In 1846, Gobley separated phospholipids from egg yolk. The term“lecithin” which is derived from the Greek lekithos was first used todescribe a sticky orange material isolated from egg yolk. “Lecithin”refers to the lipids containing phosphorus isolated from eggs andbrains; (3) from a scientific point of view, “lecithin” refers to PCs(phosphatidylcholine) the most common phospholipid, egg yolks, liver,wheat germ and peanuts contain the phospholipid lecithin.

Phospholipids (FIG. 3.0) have excellent biocompatibility. In addition,phospholipids are renowned for their amphiphilic structures. Theamphiphilicity confers phospholipids with self-assembly, emulsifying andwetting characteristics. When introduced into aqueous milieu,phospholipids self-assembly generates different super molecularstructures which are dependent on their specific properties andconditions.

In the need for synthetic analogs of natural phospholipids, furthersynthetic phospholipids were for instance designed to optimize thetargeting properties of liposomes. Examples are the PEG-ylatedphospholipids and the cationic phospholipid 1,2-diacyl-P-Oethylphosphatidylcholine. Also attempts were made to convert by organicchemical means phospholipids into pharmacological active molecules (forinstance ether phospholipids or to make phospholipid pro-drugs.

DPPC is the major constituent of stratum corneum surfactants whichcontrols the dynamic surface tension (DST) and helps maintaining theepi-dermis health. It is also one of the most popular phospholipids usedfor preparing lipid or niosome bilayers and model biological membranes.

SUMMARY

The present embodiment features methods and apparatus for producingliposomes and niosomes containing hydrophobic and hydrophilicingredients know to be beneficial to the repair and rejuvenation to thestratum corneum and underlying epi-dermis with the ability to effectnon-systemic drug absorption and transportation are influenced byvarious factors. The methods and apparatus are suitable for large scaleproduction of pharmaceutical grade liposomes which are sterile, of apredetermined size, and are substantially free of organic solvents. Thepresent embodiment features a method of making liposomes and elasticniosomes using low pressure fluids.

As constructed according to the present embodiment example, nano andmacro carriers can be either unimolecular (i.e.: dendrimers, carbonnanotubes, polymer-conjugate drug/protein, etc.) or multimolecularcarries, based on molecular self-assemblies (nanoshells, vesicles,etc.). Their major constituents are either lipids or polymers and theyall have in common that the final arrangement is governed by the natureof the initial components and the methodology used in their preparation.Some of the advantages are the incorporation of ACIs (activecosmeceutical ingredients).

One method of the example embodiment comprises the steps of forming asolution or mixture of a phospholipid, a hydrophobic or hydrophiliccosmeceutical ingredient, an aqueous phase and a low pressure fluid. Thesolution or mixture is decompressed to separate the low pressure,critical fluid, from the phospholipid and aqueous medium, to form one ormore liposomes. This method is referred to as the decompression methodof forming liposomes in the embodiment. Preferably, the rate ofdepressurization influences the size of the liposomes formed.

According to the procedure of the example embodiment, schematicallyrepresented in FIG. 4.0, operating always under mild conditions topreserve the activity of the labile biomolecules. The general methodconsists in loading a solution of the membrane lipid components and thedesired hydrophobic bio-actives in an organic solvent (e.g. ethanol),into the high-pressure reactor previously driven to the preferredworking temperature (FIG. 4.0 A). The reactor is then pressurized, in asecond stage, with a large amount of compressed CO2 until reaching theworking pressure (10 MPa) (FIG. 4.0 B).

Finally in the third stage, the vesicular conjugates are formed bydepressurizing the resulting CO2-expanded solution over an aqueousphase, which might contain water soluble surfactants and hydrophilicbio-actives (FIG. 4.0 C). In this step a flow of N2 at the workingpressure is used in order to push down the CO2-expanded solution and tokeep constant the pressure inside the reactor. It is worth to note thatno further energy input is required for achieving the desired SUVs(small unilamellar vesicles) structural characteristics, neither forincreasing the loading or functionalization.

In applications utilizing the example embodiment with low pressurefluids, the properties of the coating material and particularly theinteractions of coating materials with low pressure low temperaturefluids are especially important.

These interactions may be important for enabling the incorporation ofcosmeceutical essential oils into carrier materials, for example byfacilitating the diffusion of the essential oil due to the swelling andopening of the pores of carrier material particles.

One method comprises the steps of (1) forming a solution or mixture of aphospholipid, (2) an aqueous phase and low pressure low temperaturemethodologies. (3) The solution or mixture is decompressed to separatethe fluid, from the phospholipid and aqueous media, to form one or moreliposomes.

In some embodiments, the aqueous, or addition phase, has a therapeuticcosmeceutical agent included. As used herein, the term “therapeuticcosmeceutical agent” means a chemical or ingredient capable of effectinga desirable response in an individual subject. This embodiment isideally suited for therapeutic cosmeceutical agents which are not shearsensitive.

Preferably the compressed fluid is recycled. To the extent thatphospholipids and aqueous phase are carried over with the CF, suchcomponents may also be recycled. For convenience, liposomes formed withCF fluid in the current embodiment are referred to as “LPLTVs.”

An example embodiment features an apparatus for formingliposomes/niosomes (non-ionic) vesicles. The apparatus comprises a firstvessel wherein a phospholipid, an aqueous phase and a CF are combined toform a mixture or solution. The apparatus further comprises a secondvessel in communication with the first vessel for expansion.

The apparatus of the embodiment further comprises a third vessel fordepressurization as a means capable of reducing the pressure of thesolution or mixture. Depressurization means may be interposed betweenthe first and second vessels or may be integral with a third vessel. Thethird vessel receives the solution or mixture of phospholipids and anaqueous phase which form liposomes upon depressurization.

Preferably, the CF is removed from depressurization means and/or thethird vessel and recycled.

One example embodiment comprises the steps of forming a solution ormixture of a phospholipid and a compressed fluid. The solution ormixture is then decompressed through a tip or orifice into an aqueousphase to form one or more liposomes. As a result of the decompression,the CF is separated from the phospholipids and the aqueous phase. Thereleased CF is either vented or recycled to form a solution or mixtureof phospholipid.

A further example embodiment features a method of making liposomes orniosomes comprising the steps of forming a solution or mixture of aphospholipid and a CF. The solution or mixture is injected into anaqueous phase to form one or more liposomes or niosomes as thephospholipids and CFs are decompressed.

Preferably, the aqueous phase or phospholipids contain a cosmeceuticaltherapeutic agent which is incorporated into the liposome or niosomes.

Embodiments of the present method are ideally suited for skinrejuvenating agents which are shear sensitive such as botanicals,proteins and peptides. Embodiments of the present method do not subjectbotanicals, proteins and peptides to extreme shear forces ortemperatures.

Example embodiments are ideally suited to form unilamellar liposome orniosome vesicles. The size of the liposome or niosome is determined bythe rate of decompression.

A preferred method uses a CF selected from the group of compositionscapable of forming a critical fluid comprising carbon dioxide; nitrousoxide; halo-hydrocarbons, such as FREON; alkanes such as propane andethane; and alkanes such as ethylene.

One example embodiment features an apparatus for forming liposomes andniosomes. The apparatus comprises a first vessel for containing asolution or mixture of a phospholipid and a compressed fluid. Theapparatus further comprises a second vessel for containing an aqueousphase. The first vessel and the second vessel are in communication bymeans of injection means for injecting the phospholipid and CF fluidmixture into the aqueous phase. Upon injection into the aqueous phase inthe third vessel, liposomes are formed.

Preferably, the aqueous phase contains a cosmeceutically therapeuticagent which cosmeceutical therapeutic agent is encapsulated within theliposome.

Conjugation of cosmeceutical bio-beneficial ingredients to nano carrierscan offer over the free ingredient the protection from prematuredegradation, a higher stability, an enhance permeability throughbiological membranes, a higher control of the pharmacokinetics, a betteringredient tissue distribution profile, and an improvement ofintracellular, intercellular, and intra-follicular penetration and theability to control whether the nano-carrier goes systemic ornon-systemic.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the example embodiments, reference shouldbe made to the following detailed description disclosed in conjunctionwith the accompanying drawings, in which:

FIG. 1.0 illustrates the classification of vesicles regarding size andlamellarity.

FIG. 2.0 illustrates the construction and composition of phospholipids

FIG. 3.0 illustrates the major classifications of liposomes as vesicularsystems according to their size and membrane lamellarity.

FIG. 4.0 (A, B, C) is a representation of the steps of forming asolution or mixture of a phospholipid, an aqueous phase and low pressurelow temperature methodologies.

FIG. 5.0 is a TEM image of liposomes produced in the LPLTVs process.

FIG. 6.0 is an image of the appearance of small spheres aggregating intolarger spheres or captured within larger spheres in the LPLTVs liposomalforming process.

FIG. 7.0 shows rod or coffee-bean morphology observed in the liposomessamples produced by the LPLTVs process.

FIG. 8.0 is a schematic representation of the LPLTV process method.

FIG. 9.0 shows a solubility curve of hyaluronic acid and cholesterol, inethanol/CO2 at 10 MPa and 308 K.

FIG. 10.0 is a schematic illustration of the formation of (a) thehyaluronic acid cholesterol/CTAB bimolecular amphiphile and (b) theirself-assembling into bilayer vesicles based on the packing parameterconcept.

FIG. 11.0 is a chart showing Hyaluronic Acid levels in active andcontrol samples.

DETAILED DESCRIPTION

The present embodiment features methods and apparatus for producingcosmeceutically benevolent ingredient content liposomes and niosomes.The methods and apparatus are suitable for large scale production ofpharmaceutical and cosmeceutical grade vesicles for the treatment ofskin anomalies created as a result of aging skin or chronicenvironmental insult which are sterile, of a predetermined size, and aresubstantially free of organic solvents.

Definitions

As used herein, the word “hydrophilic” in relation to the material meansthat that material is above 10% soluble in water by weight at standardtemperature and pressure (STP).

As used herein, the word “hydrophobic” as used in relation to a materialmeans that that material is less than 0.1% soluble in water by weight atstandard temperature and pressure (STP).

As used herein, the term (IDS) as used in relation to the explanation ofthe current embodiment means Ingredient Delivery Systems.

As used herein, the word “micelle” as used in relation to a materialmeans “molecules having both polar or charged groups and non-polarregions (amphiphilic molecules) formed aggregates”.

As used herein, the word “vesicle” as used in relation to one preparedartificially, in which case they are called liposomes. If there is onlyone phospholipid bilayer, they are called unilamellar liposome vesicles;otherwise they are called multilamellar.

As used herein, the word “niosome” as used in relation to a non-ionicsurfactant-based Vesicle formed mostly by non-ionic surfactant andcholesterol incorporation as an excipient.

As used herein, the term “LPLTVs as used means Low Pressure LowTemperature alternative construction of Vesicles based on milderconditions of pressure (<10 MPa) and temperature (<308 K) than thepreviously described methodologies based on CFs (Compressed Fluids).

As used herein, the term Active Cosmetic Ingredients (ACIs) as usedmeans but is not limited to such substances as synthetic or natural skinrejuvenating ingredients, sunscreen ingredients, skin-lightening agents,and anti-acne ingredients.

As used herein, the term CFs as used means such substances made fromcompressed fluids based technologies to produce niosomes or vesicles.

As used herein, the term phospholipids as used means lipids containingphosphorus, a polar potion and non-polar potion in their structures.

As used herein, the term niosomes are microscopic lamellar vesicularstructures, which are formed on the admixture of non-ionic surfactantand cholesterol with subsequent hydration in aqueous media.

One example embodiment features an apparatus for forming liposomes andniosomes. The apparatus comprises a first vessel or mixing the organicphase, a second vessel for containing a mixture of multi-lamellarvesicles and a compressed fluid and a third vessel for decompressinginto the aqueous phase. The first vessel is in communication with asecond vessel which second vessel is in communication with a thirdvessel capable of decompressing the mixture to remove the compressedfluid. During decompression, one or more liposomes or niosomes areformed.

Another embodiment further comprises a third vessel for formingmultilamellar vesicles by hydrating phospholipids in an aqueous phase.

In the embodiment, the aqueous phase or the phospholipids may contain atherapeutic agent to impart special qualities to the liposome forbeneficial partitioning of the stratum corneum to aid in transitingcosmeceutically beneficial liposomes or niosomes to the epi-dermis.

An embodiment further features control means for determining the rate ofdecompression. The rate of decompression determines the size ofliposomes or niosomes.

Preferably, compressed fluid removed from the liposome preparation inthe decompression vessel is recycled to the first vessel to formadditional mixtures of multilamellar vesicles and compressed fluid.

Contact with compressed fluid may cause destruction of the cellularstructures particularly upon rapid decompression. Thus, embodiments are,for the most part, self-sterilizing.

Methods and apparatus of the example embodiment are capable of formingliposomes or niosomes which carry a cosmeceutical therapeutic agent. Thecosmeceutical therapeutic agent can be incorporated into ingredientswhich are used to form the liposome or niosome or the liposome orniosome can be loaded with the cosmeceutical therapeutic agent after theliposome or niosome is formed.

Embodiments allow the recovery of raw materials, lipids and solventswhich are not incorporated into the final liposome or niosome product.Example embodiments feature efficient cosmeceutical ingrediententrapment and recovery of un-encapsulated cosmeceutical ingredient. Theoperating parameters of the apparatus and method are consistent withother industrially applied processes. The method and apparatus arecapable of operating continuously.

These and other features, aspects, and advantages of the embodiment willbecome evident to those of ordinary skill in the art from a reading ofthe present disclosure.

During the depressurization step of the example embodiment, the expandedorganic solution experiences a large, abrupt and extremely homogenoustemperature decrease produced by the CO2 evaporation from the expandedsolution. This is the reason that explains the obtaining of homogenousvesicles regarding size, lamellarity and morphology compared with thesame system but prepared by a conventional mixing method.

However, changes in the procedures and equipment, as in the presentembodiment, result in vesicular systems with differentiatedcharacteristics. The processes can also be distinguished by the latterhydration step that can occur either during the pressurization or thedepressurization step.

These lipid or niosome vesicles of the present embodiment allow thephysicochemical properties of ingredient molecules, of a highermolecular weight in excess of 700 kDa, in a liposomal system to bechanged, which facilitates crossing of the stratum corneum barrier intothe epi-dermis.

The size of the liposome can be controlled by the rate of decompressionto form liposomes or niosomes of predetermined size to control thevolume and depth of penetration.

Among the various approaches for exploiting developments in nano andmicro technology for cosmetic applications, ingredient delivery systems(IDS) have already had an enormous impact on cosmetic formulationtechnology, improving the performance of many existing ingredients andenabling the use of entirely new therapies. The fact that IDSs canprotect sensitive molecules, such as hormones, enzymes and proteins,from degradation and the in-vivo attack of the immune system providinglonger resident times, have been used to improve the effectiveness anddelivery of these ingredients. Although nano and micro particulatecarriers can be made from a variety of organic and inorganic materials,vesicle and polymer based-nanocarriers are perhaps the most widely usedfor ingredient delivery purposes.

Particularly vesicles, liposome and noisome, have served as convenientdelivery vehicles for biologically active compounds because they arenon-toxic, biodegradable and non-immunogenic. Contrary to products wherethe active substance is in simple solution, the pharmacologicalproperties of vesicle-based delivery systems strongly depend on thestructural characteristics of the conjugates. Indeed, a high degree ofstructural homogeneity regarding size, morphology and vesicleorganization in the membrane is crucial, for their optimum performanceas functional entities.

Liposomes and niosomes are vesicles in which, in the current embodiment,cosmeceutical ingredients can be trapped and administered moreefficiently. However, these vesicles, micelle, liposome and niosome, arenot similar to each other. In a comparison, micelles vs. liposomes, andor elastic niosomes, the differences between the two are explained as;Micelles are structures composed of a monolayer of amphipathicmolecules. In a biological system, the molecules tend to arrangethemselves in such a manner that the inner core of these structures arehydrophobic and the outer layers are hydrophilic in nature.

Liposomes as in the present embodiment, are composed of a bilayer ofamphipathic molecules, the molecules are arranged in two concentriccircles, such that the hydrophilic heads of the outer layer are exposedto the outer environment, and the hydrophilic heads of the inner layermake the inner hydrophilic core. The hydrophobic tails are tuckedbetween the two layers.

In the present embodiment, elastic liposomes are microscopic vesicleshaving single or multiple phospholipid bilayers which can entraphydrophilic compounds within their aqueous cores.

Elastic niosomes are composed of nonionic surfactants, ethanol andwater. They are superior to conventional niosomes because they enhancepenetration of a drug through intact skin by passing through pores inthe stratum corneum, which are smaller than the vesicles. In fact, theirelasticity allows them to pass through channels that are less than onetenth of their own diameter. Thus they can deliver ingredients orcompounds of both low and high molecular weight. Furthermore, they canprovide prolonged action and demonstrate superior biological activitycompared to conventional niosomes. The transport of these elasticvesicles is concentration independent and driven by trans-epidermalhydration.

To deliver the molecules to sites of action, the lipid or niosomebilayer can fuse with other bilayers such as the cell membrane, thusdelivering the liposome contents. By making liposomes in a solution ofnatural or synthetic ingredients that can effect a beneficial change tothe skin, (which would normally be unable to diffuse through themembrane) they can be (indiscriminately) delivered past the lipidbilayer. A liposome or niosome vesicle does not necessarily havelipophobic contents, such as water, although, in the case of the presentembodiment, it usually does.

The preferred phospholipid in the current process embodiment isnaturally derived, for example phospholipids obtained from plant oranimal sources. Natural phospholipids are purified from, e.g., soybean,rapeseed, and sunflower seed. The phospholipid may be salted ordesalted, hydrogenated or partially hydrogenated or natural,semi-synthetic or synthetic.

Liposomes, niosomes and in general vesicles, are undoubtedly one of themost promising carriers in nano and micro cosmeceutical ingredientdelivery. They are particularly important in the stratum corneumpercutaneous transit field due to their great versatility respect tosize, composition, surface characteristics, biocompatibility,biodegradability, low toxicity, capacity for entrapping and/orintegrating hydrophilic and/or hydrophobic molecules and possibility ofsurface functionalization. Vesicles of the present process embodimentare spherical objects enclosing a liquid compartment, with a diameterranging from 20 nm to a few thousand of nanometers, separated from itssurroundings by at least one thin membrane consisting of a bilayer(unilamellar) or several layers (multilamellar) of amphiphilicmolecules.

Sometimes the terms liposome, niosome and vesicle are usedinterchangeably, although a liposome is a type of vesicle composedmainly by phospholipids, a niosome as a non-ionic surfactant-basedvesicle formed mostly by non-ionic surfactant and cholesterolincorporation as an excipient. Vesicles can be formed also by non-lipidbuilding blocks, such as block co-polymers or cationic or non-ionicsurfactants.

A liposome or niosome is an artificially-prepared vesicle composed of alipid bilayer. The liposome or niosome can be used as a vehicle foradministration of percutaneous skin nutrients and pharmaceutical drugs.Liposomes and niosomes are composed of natural phospholipids, and mayalso contain mixed lipid chains with surfactant properties (e.g., eggphosphatidylethanolamine). According to the present process embodiment,a liposome design may employ surface ligands for attaching to unhealthytissue.

In the present embodiment, phospholipids have a propensity to formliposomes and niosomes, which can be employed as the cosmetic ingredientcarriers. Phospholipids have good emulsifying property which canstabilize the cosmetic serum emulsions. In addition, phospholipids assurface-active wetting agents which can coat on the surface of crystalsto enhance the hydrophilicity of hydrophobic ingredients. The aboveproperties are successfully employed in the LPLTVs design.

As used herein, in the current embodiment example, the term“phospholipid” refers to compositions which are esters of fatty acids inwhich the alcohol component of the molecule contains a phosphate groupas an integral part (FIG. 2.0).

In order to extend LPLTVs (Low Pressure Low Temperature alternativeconstruction of Vesicles) to the preparation of other kinds of vesiclesystems taking full advantage of the possibilities offered by thisprocess were also undertaken. Phospholipids-based formulations arewidely used for delivery purposes and for this reason1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was selected as amembrane component for the liposome preparation with LPLTVs.

Phospholipids comprise the glycerol-phosphatides, containing glycerol,and the sphingomyelins containing sphingosine.

According to the alcohols contained in the phospholipids, they can bedivided into glycerophospholipids and sphingomyelins.

For the present embodiment, the use of Glycerophospholipids, which arethe main phospholipids in eukaryotic cells, refer to the phospholipidsin which glycerol is the backbone are preferred. All naturally occurringglycerophospholipids possess α-structure and L-configuration.

Preferred phospholipids used in the embodiment comprisephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine andsphingomyelin; and although not preferred, in the present embodiment,synthetic phospholipids comprising dimyristoyl phosphatidylcholine,dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine,distearoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol,dimyristoyl phosphatidylserine, distearoyl phosphatidylserine, anddipalmitoyl serine.

In the case of the present method embodiment, liposomes or niosomes areused as carriers for beneficial ingredients for the treatment of skinanomalies. Liposomes and niosomes can be made with different featurescan enhance an ingredients efficacy, reduce an ingredients toxicity,restriction from going systemic and prolong the ingredients therapeuticeffect.

Niosomes are self-assembled vesicles composed primarily of syntheticsurfactants and cholesterol. They are analogous in structure to the morewidely studied liposomes formed from biologically derived phospholipids.

The type of epi-dermal activity resulting from the application of thecurrent embodiment's content of natural or synthetic ingredients to bebeneficial includes: Hydration •Skin lightening •Anti-wrinkle/skinsmoothing •Antioxidant activity/free radical scavenger•Anti-inflammatory/anti-irritant •Collagen stimulation •Cellregeneration/stimulation •Sebum regulation •Anti-cellulite•Antimicrobial •Antibacterial.

With chronological age and chronic exposure to adverse environmentalfactors, (notably UVA, UVB, and IR radiation) the visual appearance,physical properties, and physiological functions of skin change in waysthat are considered cosmetically undesirable. The most notable andobvious changes include the development of fine lines and wrinkles, lossof elasticity, increased sagging, loss of firmness, loss of colorevenness (tone), coarse surface texture, and mottled pigmentation.

Less obvious, but measurable changes which occur as skin ages or endureschronic environmental insult include a general reduction in cellular andtissue vitality, reduction in cell replication rates, reduced cutaneousblood flow, reduced moisture content, accumulated errors in structureand function, and a reduction in the skin's ability to remodel andrepair itself.

Many of the above alterations in appearance and function are caused bychanges in the outer epidermal layer of the skin, while others arecaused by changes in the lower dermis.

Regardless of the stimulus for skin damage, when damage occurs, numerousnatural and complex biochemical mechanisms are set into motion inattempts to repair the damage.

The present embodiment relates generally to construct a process for avesicle-driven treatment method and composition for improving the skin'svisual appearance, function, and clinical/biophysical properties whichhave been changed by factors such as chronological age, chronic sunexposure, adverse environmental pollutants, household chemicals, diseasepathologies, smoking, and malnutrition. In particular, the presentembodiment relates to a process to create a method of treating skin byincreasing the skin's stratum corneum transit of known beneficialingredients through dynamic infusion of vesicles (DIV) generated fromnatural and biocompatible phospholipids with an aqueous volume enclosedwithin a lipid or niosome membrane.

The result of the present process embodiment is to deliver largermolecular weight, longer lasting, beneficial ingredients to areas of theepi-dermis depleted of needed vitamins, hydration, nourishment andcomplimentary ingredients need for the rejuvenation of elastin andcollagen.

Now, in the current embodiment, comes the development of a new, singleprocess, ingredient vesicle methodology based on a Low Pressure LowTemperature alternative construction of liposome or niosome Vesiclesprocess (LPLTVs) for the direct, robust and scalable encapsulation ofbiomolecules in vesicles. The development of reproducible and scalablemethodologies in order to functionalize those vesicles withtargeting/protective units enabling greater selectivity of thetherapeutic epidermal targets and therefore more effective treatments.

The use of the biomolecules-vesicles conjugates prepared by LPLTVs canbe used in the treatment of different skin anomalies. The embodimentsprocess uses milder conditions of pressure (<10 MPa) and temperature(<308 K) than previous methodologies based on CFs, allowing theprocessing of heat labile compounds and reducing the investment cost ofa high pressure plant when the process is scaled-up.

The present embodiment encompasses compressed fluid-based methodologies(CF), also called dense gas technologies, for the production oflipid-based ingredient carrier systems with structural characteristicsnot reachable by already existing procedures using liquid organicsolvents. In the present embodiment, we have improved the processing ofvesicles and niosomes because they provide the ability to reduce theamount of organic solvent required by conventional methods and allow abetter control over the final vesicle structural characteristics.Moreover compressed fluid processing offers sterile operating conditionsand the ability for one-step production processes, which is convenientin transferring the technology to larger scale operations.

The present embodiment's compressed fluid technology was developed as aplatform for producing lipid and niosome-based cosmetic ingredientcarrier systems that can address most of the limitations of conventionalmethods.

LPLTVs methodology allows an easy and direct preparation of differentliposome-biomolecule conjugates with micro and nano scopic sizes andgreat degrees of unilamelarity.

The stability time of the liposome-based conjugates is somewhat smallerthan those of LPLT Vesicle-based conjugates. This stability is improvedby the addition of stabilizing/targeting units to the formulation.

Bioactivity of the integrated biomolecules is unaffected under theprocessing conditions with CO2-expanded solvents.

Liposomes and Niosomes prepared by the current embodiment's process ofLPLTVs, fulfill the structural and physio-chemical requirements to be aplatform for the percutaneous delivery of synthetic or natural ACIs(active cosmeceutical ingredients).

Major advantages of the embodiment's application of CFs technology arethat sterile and stable liposomal and niosomal formulations can beproduced with minimum amounts of organic solvents.

In the case of blemished or compromised complexion of the skin thefollowing properties could be desirable: •Sebum regulating•Anti-bacterial •Anti-inflammatory/anti-irritant •Soothing/calming •Skinhealing and regeneration •uniform complexion •lightening andbrightening.

These and other advantages will be apparent to individuals skilled inthe art in view of the drawings and detailed description which follow.

Examples of some preferred preparation ingredients in the presentembodiment include natural botanicals, those ingredients that thatoriginates from plants, herbs, roots, flowers, fruits, leaves or seedssuch as: aloe vera, almond oil, avocado oil, coconut oil, hazelnut oil,jojoba oil, olive oil, palm oil, pumpkin seed oil, sesame oil, sunfloweroil, tamanu oil, candeia oil, arnica,

chamomile, oat extract, hibiscus flower, boswellia serratta, cocoapowder, green and white tea, gotu kola, chamomile extract, L-arginine,glutamine, pantothenic acid, white willow bark extract,tetrahydrocurcuminoids, alpha-arbutin, aloesin, alpha glucosylhesperidin, niacinimide, fucoidan, magnesium asorbyl phosphate, azelaicacid, N-acetyl-D-glucosamine, glutathione, mulberry, pomegranate seedoil, cyprus rotund root extract, licorice, licorice-glabrin rootextract, kojic acid, panax ginseng root extract, ginko bilbao, salicylicacid, Lauric acid, glycerin, caffeine, tocopheryl acetate, copperpeptide, retinyl palmitate, asorbyl palmitate, wakame,dimethylethanolamine, beta glucan, triglyceride as well as hyaluronicacid (Hyaluronic acid is a natural and sugar-like biopolymer in thehuman body that alternately consists of D-glucuronic acid andN-acetyl-D-glucosamine-units).

Additionally, preferred natural polymers for the current embodiment suchas starch, starch, xanthan or guar gum, carrageenan, alginates,polysaccharides, pectin, gelatin, agar, and cellulose derivatives can beused to this end. On the synthetic side, polyacrylate derivatives andpolyacrylamide polymers can be incorporated in to the carrier system ofthe present embodiment. More recent developments include combininghydrophobic and hydrophilic polymers into block and star copolymers andthermally responsive systems.

Polymers are particularly susceptible to the construction of vesiclethat can physically entrap the active component, preserving itsbiological stability, or the bioactive component can be incorporatedchemically into a polymer chain or pendant group, then released throughhydrolysis. For example, salicylic acid (an anti-acne ingredient) can beincorporated into the main chain of polyanhydride ester and releasedwithin a short time.

The current example embodiment also applies to the construction ofvesicle encapsulated polymers that are routinely used in many personalcare and cosmetic products.

The current embodiment takes advantage of the various properties ofthese polymers to impart unique benefits to their formulations. Therange of properties is as varied as the class of polymers that have beenutilized. Using polymers, cosmetic chemists can create high performanceproducts. Broad spectrums of polymers; natural polymers, syntheticpolymers, organic polymers as well as silicones are used in a wide rangeof cosmetic and personal care products as film-formers, emulsifiers,thickeners, modifiers, protective barriers, and as aesthetic enhancers.

A further embodiment features a method of making liposomes comprisingforming a mixture of multilamellar vesicles and a CF. The mixture isdecompressed to remove the CF to form one or more liposomes or niosomes.

Preferably, multilamellar vesicles are made by hydrating phospholipidsin an aqueous phase. Preferably, the aqueous phase or the phospholipidscontain a cosmeceutical therapeutic agent.

EXAMPLE 1 Phase Behavior Studies for the Low Pressure Low TemperatureAlternative Construction of a Liposome and Niosome Vesicles(LPLTVs)—CO2-Solvent System

Prior to liposome or niosome formation, the phase behavior andsolubility of the chosen lipid in dense CO2 were investigated to verifythe suitability of the lipid for dense gas processing and, in particularLPLTVs processing.

Knowledge of the threshold pressure for precipitation of lipid fromsolution is also a key factor for design of the LPLTVs process in orderto determine the maximum pressure for the technique so that yield isenhanced and loss of lipid in the expansion chamber minimized. The solidstate of 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) wasmaintained when the lipid was exposed to CO2 below 350 bar at 50° C. and150 bar at 70° C. The solubility of DSPC in pure CO2 at 50° C. andpressures up to 280 bar was considered negligible. The addition of 5 mol% ethanol co-solvent did not significantly improve the solubility ofDSPC in CO2 at 50° C. and 250 bar. Use of higher pressures or largeramounts of organic solvent are undesirable, thus the results of thesolubility study are in agreement with the literature in concluding thateffects arising from poor solubility of lipids in dense CO2 are noteasily overcome.

In prior art, at 50° C. and 250 bar, DSPC required the addition of 4.8%v/v ethanol as well as the use of a recycling system for homogeneousdissolution of the lipid in CO2. In the current embodiment, the use ofthe LPLTVs process eliminates the current limitations of dense gastechniques associated with solubilizing lipids using a supercriticalfluid and simply utilizes a dense gas as an aerosolization aid. Thethreshold pressure for the precipitation of DSPC from a 10 mg/mL ethanolsolution at 22° C. was 55 bar.

Precipitation was first observed at 58, 55, and 56 bar for the 5, 10,and 20 mg/mL solutions of DSPC and cholesterol (70:30 lipid tocholesterol weight ratio) in ethanol at 22° C., respectively. Thereforeit can be seen that cholesterol had negligible effect on the thresholdpressure. When the pressurization rate for the 5 mg/mL lipid/cholesterolsolution was dramatically increased, precipitation was not observeduntil 60 bar was reached. A faster pressurization rate is preferable forthe embodiments LPLTVs process in order to minimize the time requirementfor each experiment. During this experiment, noticeable expansion onlystarted to occur after 50 bar was reached. Solution expansion is desiredto maximize the effect of utilizing CO2 as an aerosolization aid todisperse the lipid solutions throughout the aqueous phase. Therefore,the expansion pressure used in the LPLTVs experiments to avoid soluteprecipitation and enhance the yield for or niosome formation fromethanol solutions was between 50 and 55 bar at 22° C.

The threshold pressure for the precipitation of a 20 mg/mLDSPC/cholesterol chloroform solution (90:10 lipid/cholesterol weightratio) at 22° C. was 41 bar. The solvent volume had significantlyexpanded (doubled) by the time 40 bar was reached in the chloroformexperiments. Therefore, expansion pressures between 38 and 40 bar wereused for the LPLTVs chloroform experiments to achieve maximum expansionwithout lipid precipitation.

EXAMPLE 2 Effects of Process Variables on LPLTVs Operation

The effects of solute composition, solute concentration, type ofsolvent, nozzle diameter, type of aqueous media, temperature of vesicleformation chamber, and volume of dense gas used for spraying on both theease of operation of the embodiments LPLTVs process and the product wereinvestigated. The results obtained for liposome formation are summarizedin Table 1. Preliminary trials were conducted to establish viable nozzleoptions for the LPLTVs system. A variety of nozzles were testedincluding 102, 178, 254, 508, and 1016 μm i.d. stainless steel tubingand 100 μm i.d. Peeksil tubing (polymer tubing with fused silicalining). The most suitable nozzle for the LPLTVs apparatus, to controlthe flow rate and prevent blockages, was the 178 μm i.d. stainless steeltubing. The 254 μm nozzle was used in Set 1 (Table 1); however, therewere difficulties in controlling the flow rate and maintaining constantpressure in the expansion chamber. Other nozzle dimensions may beselected depending on the pump capacity and vessel dimensions.

The LPLTVs process of the present example embodiment is robust and,within the range examined, variation of solute concentration andcomposition, type of solvent, type of aqueous media, and volume of CO2used for spraying had minimal effect on the operation of the LPLTVsprocess. The temperature of the vesicle formation chamber did, however,significantly affect the process since a smaller amount of liposomalproduct was obtained at 90° C. (Set 5) compared with 75° C. The smallervolume can be attributed to the aqueous medium being closer to itsboiling point at 90° C., and thus some of the water was lost to thesolvent trap via evaporation.

EXAMPLE 3 Characterization of Liposomes Produced by the LPLTVs Process

The Liposome Morphology.

TEM (Transmission electron microscopy) was used to investigate themorphology of the particles produced in the embodiments LPLTVs process.At all conditions studied, submicron spheres were observed thatpossessed a similar structure to liposomes previously reported in theliterature. The image shown in FIG. 5.0 indicates that sphericalparticles, generally ranging in size from 35 to 200 nm and more commonly35-100 nm, were formed using the LPLTVs process. Images collectedsuggest that the liposomes were unilamellar. Not only were the spheresof a size range common to unilamellar liposomes, but in many images asingle, thin wall can be seen at the edge of each particle. However, thearguments against positive identification of lamellarity using negativestaining and TEM have been well documented in the literature.18 Stainingartifacts are difficult to identify and are often interpreted asunexpected morphologies. Confirmation that the particles formed were infact liposomes was found by utilizing SANS (Small-Angle NeutronScattering) to identify an aqueous core, as discussed below. Thespherical particles shown in FIG. 5.0 are a general indication of theliposomes formed; however, some other features have also been observed.In several samples, a large quantity of smaller spherical particles(10-20 nm) was observed, which are at or below the lower size limit atwhich liposomes can be formed and may be considered as micelles. In somesamples, small vesicles appear to be aggregated into or contained withina larger liposome vesicle, as shown in FIG. 6.0.

A vesicle-in-vesicle structure may be formed in the last stage of theLPLTVs process due to liposomes forming in the presence of existingvesicles. However, the lipid vesicles are more likely to have formedinto aggregate structures during the negative staining process in orderto minimize any deleterious effects when the aqueous phase was removedor to minimize the interactions of the lipid with the stain. Theartifact of these aggregated systems could also result from a largervesicle superimposed upon smaller vesicles, which is a common feature inTEM analysis. The particle size and morphology of the LPLTVs liposomeswas not significantly changed within the range of process parametersvaried. However, rods or coffee bean morphology (liposomes exhibiting acharacteristic ‘coffee-bean’ appearance due to the presence of an innerstructure apparently separating the LUV into two sections) appeared in afew samples in addition to spherical particles, as shown in FIG. 7.0. Itis suggested that the coffee bean morphology was formed due to thecollapse of vesicles, predominantly for the smaller particles. Thiseffect can be attributed to the lower stability of small vesicles due tothe high curvature of the membrane.

The unilamellar liposomes produced using a conventional technique werestained with ammonium molybdate with and without the presence ofprotein. The images of the liposomes stained without protein showed“cup-like structures” and vesicles consisting of two lipid membranes.When protein was included in the staining process, the images showedvesicles consisting of a single lipid bilayer.

In the present embodiment, it is concluded that the liposomes in bothimages were unilamellar and that the vesicles had collapsed in theabsence of protein. The double membrane feature can therefore beexplained by the thick edge of the collapsed sphere, and the “cup-likestructures” can be observed if the collapsed spheres are rotated.

The correct choice of vesicle or niosome preparation method in thecurrent embodiment depends on the following parameters: thephysicochemical characteristics of the material to be entrapped andthose of the liposomal or niosomal ingredients; the nature of the mediumin which the vesicles are dispersed; the effective concentration of theentrapped substance and its potential toxicity; additional processesinvolved during application/delivery of the vesicles; optimum size,polydispersity and shelf-life of the vesicles for the intendedapplication; and, batch-to-batch reproducibility and possibility oflarge-scale production of safe and efficient liposomal products.

TABLE 1 Summary of the Conditions Investigated and the Results Obtainedfor Producing Liposomes via the embodiments of the LPLTVs Process. set 12 3 4 5 6 7 8 9 nozzle diameter (μm) 254 178 178 178 178 178 178 178 178solute lipid content 70 70 90 90 90 90 90 90 90 (% w/w) solute conc.(mg/mL) 20 20 20 5 20 20 20 20 20 VFC temp. (° C. ± 2.5) 75 75 75 75 9075 75 75 75 CO₂ spraying vol. (mL) 200 200 200 200 200 50 200 200 200aqueous media RO H₂O RO H₂O RO H₂O RO H₂O RO H₂O RO H₂O DI H₂O TBS ROH₂O organic solvent EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH Chlfmeffective diameter (nm) 156 ± 2    166 ± 5    121 ± 1    162 ± 23  207 ±75  122 ± 2    119 ± 3    143 ± 5    387 ± 64  polydisperity 0.27 ± 0.010.27 ± 0.01 0.15 ± 0.01 0.19 ± 0.01 0.18 ± 0.01 0.15 ± 0.01 0.17 ± 0.020.18 ± 0.02 0.29 ± 0.03 product lipid content 75.2 ± 1.9  77.3 ± 1.8 82.4 ± 0.4  76.1 ± 0.1  81.8 ± 0.5  80.5 ± 0.2  80.9 ± 0.3  80.1 ± 0.6 81.8 ± 1.3  (% w/w) residual solvent 3.1 ± 0.4 1.6 ± 0.8 1.4 ± 0.4 2.2 ±0.3 1.9 ± 0.7 3.9 ± 0.2 1.8 ± 0.4 2.0 ± 0.8 0.4 ± 0.3 (% v/v) *VFC:vesicle formation chamber, RO H₂O: water purified via reverse osmosis;DI H₂O: deionized water; TBS: TRIS buffered saline; EtOH: ethanol;Chlfm: chloroform

Collapsed spheres were present in all LPLTVs samples; however, rods or“coffee bean” particles were rare except in those samples from Sets 1and 2, where a higher proportion of cholesterol was used compared withother samples.

TABLE 2 The calculated SLD for the components LPLT Vs of the LPLT Vsliposome samples material SLD (× 10⁻⁶ Å⁻²) H₂O −0.56 D₂O 0.33hydrocarbon Chain (CH₂)_(n) −0.44 cholesterol (C₂₇H₄₅OH) 0.21 lipidheadgroup (C₁₀H₁₃O₈NP) 1.12

TABLE 3 SANS Fitted parameters for the LPLT liposome sample fittedparameter (Å⁻²) value Core SLD 6.30 × 10⁻⁶ Shell SLD 3.85 × 10⁻⁶ SolventSLD 6.82 × 10⁻⁶

Cholesterol was incorporated in order to improve stability, and it hasbeen reported in the literature that the incorporation of cholesterolcauses larger liposomes or niosomes to form.

However, the rod-shaped particles were at the smaller end of the sizerange for the LPLTVs. Comparison of images from a number of samplesindicated that the presence of rods may be promoted by the level ofstain as well as the size of the vesicles. It is therefore also possiblethat the relative proportion of rods found in Sets 1 and 2 was amplifiedby the staining process. Because of the improved spherical morphologyobserved in Set 3, the experiments were carried out using a preferredlipid/cholesterol ratio of 90:10.

Advantages of the Current Embodiments LPLTVs Process for Bulk Liposomeor Niosome Vesicle Formation.

The LPLTVs process has many advantages over conventional liposome orniosome formation techniques. These advantages include the fact that itis a simple and rapid process for bulk production of unilamellarliposomes or niosomes. A conventional liposome standard was produced,and the formation process took almost 24 h and multiple stages tocomplete. The embodiments LPLTVs process produced a greater volume ofthe same formulation in less than half an hour, clearly demonstratingthe dramatic reduction in processing time.

The conventional ethanol and ether injection methods exhibit somesimilarities to the embodiments LPLTVs process since they involve thedissolution of a lipid into an organic phase, followed by the injectionof the lipid solution into aqueous media forming liposomes. Thedrawbacks of the ethanol injection method as opposed to the examples ofthe present embodiment, are the poor homogeneity of the vesicles ifthere is not adequate mixing and the residual solvent levels in theproduct.

Either injection method eliminates the residual solvent issue by havinga heated aqueous phase, but is a time-consuming technique. It has beensuggested that injecting the ether solution at a rate faster than 0.2mL/min can cause cooling of the aqueous phase due to evaporation, andthat pre-evaporation of ether can cause nozzle blockages and theformation of multilamellar vesicles. The LPLTVs process of the presentembodiment for the formation of liposomes or niosomes formed aroundcosmeceutically benevolent ingredients has significant advantages overboth the ethanol and ether injection methods since the depressurizationfrom a high pressure environment creates outstanding dispersion of thelipid solution and mixing with the aqueous environment. Theincorporation of both heating and dense gas washing enables the solventto be efficiently removed. The LPLTVs process can also produce anequivalent volume of product in a significantly reduced time span.Compared with other dense gas processes developed for liposomeformation, the LPLTVs process is beneficial due to its simplicity andthe incorporation of residual solvent removal measures into the method.The LPLTVs process also operates at pressures generally less than 60 barand moderate temperatures, therefore making the process morecost-effective and avoiding the concerns of uncontrollable foamformation present in the low pressure liposome method. A significantadvantage of the LPLTVs process is that it can be used to process abroad range of materials since there is no requirement for the compoundto be solubilized in the dense gas and there are no high shear forces.Furthermore, time-consuming solubility studies and recycling loops forlipid solubilization are not needed. The only preliminary investigationrequired is the determination of the threshold pressure forprecipitation of the solutes from expanded solution, such that thesolution expansion can be carried out without precipitation.

In the LPLTVs process of the current embodiment, the entrapment ofhydrophilic compounds may be achieved through the dissolution of thetarget compound into the aqueous media prior to release of the lipidsolution. The liposomes or niosomes would then form, entrapping thehydrophilic or hydrophobic compound within the aqueous interior of thevesicle.

To entrap a hydrophobic, hydrophilic, lipophilic, or amphipathiccompound into liposomes or niosomes using the LPLTVs process, thecompound is dissolved along with the phospholipid and other solutes inthe liquid solvent.

The compound then becomes entrapped within the phospholipid membrane asa result of the affinity of the compound for the membrane rather thanthe aqueous phase.

The suitability of the LPLTVs process for entrapping hydrophobiccompounds has already been demonstrated through incorporating up to 25%w/w cholesterol into the liposome formulation. The LPLTVs technique canalso be applied to the formation of structures other than liposomes.Micro particles of hydrophobic compounds could be produced throughprecipitation into aqueous media in the LPLTVs process.

Liposomal Particle Size Distribution and Stability. Photon correlationspectroscopy (PCS) was used to assess the particle size distribution ofthe liposomal population using the Brookhaven ZetaPlus. Each liposomalsample was diluted in RO or DI water and placed in a disposablepolypropylene cuvette. Ten runs, each of 1 min duration, were conductedat 23-25° C. for each sample. A laser wavelength of 678 nm was used witha destination angle of 90°. The dust cutoff was set between 20 and 50μm. The instrument calculates an effective diameter for each run and anoverall effective diameter for the 10 runs combined. The effectivediameter is the mean diameter that is calculated by the followingequation:

${{Effective}\mspace{14mu}{diameter}} = {\left( \frac{1}{d_{k}} \right)^{- 1} = \frac{\sum\limits_{i}{N_{i}d_{i}^{6}P_{i}}}{\sum\limits_{i}{N_{i}d_{i}^{5}P_{i}}}}$

Where Ni refers to the number per scattering volume of the ith particle,and Pi accounts for angular scattering effect for particles larger thanλ/20. Pi is calculated using Mie theory and requires the particlerefractive index; however, for Rayleigh scatters and at sufficiently lowangles, Pi=1 is used in the program.

EXAMPLE 4 A Formulation for the Treatment of Acne Made Using the CurrentLPLTVs Embodiment

A solution for treating Acne vulgaris or Propionibacterium acnescontaining lipids formed of the following ingredients utilizing thescience of the present embodiment may be formulated using theconstructed phospholipids of the following volumes;

D.I. water 50% to 95% (preferably 60 to 90%, ethanol 15 to 40%(preferably 25 to 30%), hyaluronic acid 5 to 50% (preferably 12 to 18%)propanediol 10 to 80% (preferably 20 to 25%), aloe vera 0.2 to 20%(preferably 0.5 to 5%), azelaic acid 2 to 50% (preferably 4 to 8%),salicylic acid 0.2 to 20% (preferably 0.5 to 5.0%), lauric acid 0.2 to20% (preferably 0.5 to 5.0%), asorbyl palmitate 0.1 to 20% (preferably0.2 to 8%) niacinimide 0.2 to 20.0% (preferably 0.5 to 5%), lecithin 0.2to 10% (preferably 0.5 to 5.0%), glycerin 0.5 to 25% (preferably 2 to10%), caffeine 0.2 to 20% (preferably 0.5 to 10%)

EXAMPLE 5 A Formulation for the Enhanced Hydration and the Reduction ofFine Lines and Wrinkles Made Using the Current Embodiment

A solution for treatment of lack of skin hydration and the reduction offine lines and wrinkles containing lipids formed of the followingingredients utilizing the science of the present embodiment may beformulated using the constructed phospholipids of the following volumes;D.I. water 50% to 95% (preferably 60 to 90% ethanol 15 to 40%(preferably 25 to 30%), hyaluronic acid 5 to 50% (preferably 12 to 18%)propanediol 10 to 80% (preferably 20 to 25%), aloe vera 0.2 to 20%(preferably 0.5 to 5%), hexa-peptide 8 2% to 50% (preferably 5% to 20%),caffeine 0.2 to 20% (preferably 0.5 to 10%), glycerin 0.5 to 25%(preferably 2 to 10%), tocopheryl acetate 0.1 to 10% (preferably 0.5 to8%), retinyl palmitate 0.1 to 10% (preferably 0.5 to 8%), asorbylpalmitate 0.1 to 20% (preferably 0.2 to 8%), Copper tri-peptide GHK-Cu0.1 to 20% (preferably 0.2 to 8%), hesperidin 0.1 to 20% (preferably 0.2to 8%), dimethylethanolamine (DMAE) 0.05 to 20% (preferably 0.08 to 8%),sesame oil 2 to 50% (preferably 3 to 20%) beta glucan 0.1 to 20%(preferably 0.2 to 8%)

TEST 1 LPLTVs Method for the Preparation of Hyaluronic Acid-richVesicles

The present embodiment is based on the use of compressed CO2 in aprocess called LPLTVs for the production of micron-sized andsubmicron-sized crystalline particles from an organic solution. Asnovelty the process used the CO2 as co-solvent being completely miscibleat a given pressure and temperature with a specific solution of anorganic solvent containing the solute to be crystallized. In order totake full advantage of compressed fluid processing without using severeworking conditions a novel and improved procedure based on the LPLTVsprocess was developed. This method, named as LPLTVs (Low Pressure LowTemperature alternative construction of Liposome Vesicles), enabled thepreparation of cholesterol rich-hyaluronic acid vesicles. The processuses milder conditions of pressure (<10 MPa) and temperature (<308 K)than the previously described methodologies based on CFs, allowing theprocessing of heat labile compounds and reducing the investment cost ofa high pressure plant when the process is scale-up. Using thisprocedure, homogeneous nanovesicles composed of hyaluronic acid,cholesterol and the cationic surfactant CTAB (cetyltrimethylammoniumbromide, in a molar ratio 1:1, were prepared by depressurizing avolumetric expanded organic solution containing the cholesterol andhyaluronic acid over a flow of an aqueous solution containing the CTABsurfactant (FIG. 5.0). An alternate non-ionically formed elastic noisomecan be constructed using the same apparatus.

During the depressurization step, the expanded organic solutionexperiences a large, abrupt and extremely homogenous temperaturedecrease produced by the CO2 evaporation from the expanded solution.This explains the obtaining of homogenous vesicles regarding size,lamellarity and morphology.

In order to prepare any vesicular system using LPLTVs is necessary thatthe lipids forming the membrane are completely soluble in theCO2-expanded organic solvent, presenting one phase at the workingconditions of pressure, P w, temperature, T w and CO2 molar fraction,X2. Therefore for the preparation of cholesterol rich-hyaluronic acidvesicles by LPLTVs method is always necessary to analyze the solubilitybehavior of the used sterol in CO2-expanded solvents, by means of adetailed phase diagram study, like the one showed in FIG. 6.0.

An important prerequisite for the effective use of vesicles as acosmeceutical ingredient carrier as described above is to control theirstability, which can be defined as the extent to which the carrierretains its ingredient contents either in vitro or in vivo studies. Oneof the major disadvantages when using classical vesicles based onphospholipids, is the leakage of the encapsulated ingredient duringtheir storage. One variant that can enhance the retention of drugs andpromote the stability of liposomes or niosomes is the presence ofhyaluronic acid in the formulation. Another variant is the preparationof liposomes from non-phospholipid amphiphiles, such as surfactants orpolymers.

This kind of vesicular formulations show low passive leakage incomparison to liposomal systems based only on phospholipids andtherefore a higher retention of the encapsulated materials, as forexample epi-dermal therapeutically active molecules.

In the present example embodiment for the preparation of positivelycharged vesicles composed by cholesterol, hyaluronic acid and thecationic surfactant hexadecyltrimethylammonium bromide (CTAB). Morerecently nanoscopic vesicles, composed by different sterols and otherquaternary ammonium surfactants have been also successfully prepared.This is why it was decided to name this kind of formulations as LPLTV(low pressure low temperature vesicles) that are stable for periods aslong as several years, their morphology do not change upon rising thetemperature or by dilution and they show a great homogeneity regardingsize and morphology.

Studies at molecular level of the self-assembling of cholesterolhyaluronic acid and CTAB molecule in aqueous medium showed that a purevesicular phase is only formed at equimolar ratios of both components.Moreover molecular dynamic (MD) simulations revealed that thecholesterol, hyaluronic acid and the CTAB self-assemble in a uniquebimolecular synthon that can be considered as a single entity whichfurther self-assembles in particularly stable vesicles (FIG. 7.0) (FIG.10). Moreover, MD simulations have provided a theoretical support tojustify the experimental high thermal stability and the exceptionalmorphological properties attributed to cholesterol, hyaluronic acid/CTABvesicles at 1:1 molar ratio.

TEST 2 Analysis of Active and Placebo Tape Strips from an In-Vivo Studyof Skin Permeation of 800 KDa Hyaluronic Acid Using the EmbodimentsLPLTVs Formation Process

Introduction

The test was to extract and analyze tape strips and blanks from an invivo tape stripping study of 800 KDa Hyaluronic Acid skin penetrationtransport.

HA distribution in stratum corneum (SC) layer was investigated.Distribution in SC was studied using a tape-stripping method.

Methods:

Each tape sample will be extracted individually by extraction solvent:1×PBS with 0.2% NaN3/acetonitrile (50/50 v). Samples with extractionsolvent were vortexed at high speed for 1 minute followed withcentrifugation at 12,000 rpm for 10 minutes (chill the samples on ice at4° C. and then centrifuge). The supernatant solution was then collectedfrom each tube/container and stored at 4° C. and ready for analysis.

Outcome:

See the chart in FIG. 11.0 showing Hyaluronic Acid levels in active andcontrol samples.

Abstract of the Disclosure is provided to allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the claims. In addition, in the foregoing DetailedDescription, it can be seen that various features are grouped togetherin a single embodiment for the purpose of streamlining the disclosure.This method of disclosure is not to be interpreted as reflecting anintention that the claimed embodiments require more features than areexpressly recited in each claim. Rather, as the following claimsreflect, inventive subject matter lies in less than all features of asingle disclosed embodiment. Thus, the following claims are herebyincorporated into the Detailed Description, with each claim standing onits own as a separate embodiment.

What is claimed is:
 1. A method for the construction of cosmeceuticallybioactive compositions including elastic niosome vesicle epi-dermaldelivery vehicles without use of supercritical CO₂ and in a continuouslyoperating process, the method comprising: forming a liposome solution ormixture of cosmeceutically benevolent phospholipids, the solution beinghydrophobic or hydrophilic, the solution including an ingredient fromthe group consisting of a natural ingredient and a synthetic ingredient,the solution having an aqueous phase; removing any constituentattributes of water-insolubility from the solution, while operatingunder conditions to preserve activity of labile biomolecules; loadingthe liposome solution and desired hydrophobic bio-actives into anorganic solvent residing in a pressure reactor, the pressure reactorhaving been previously driven to a working temperature less than 31.1°C.; pressurizing the reactor with compressed CO₂ until reaching aworking pressure less than 73.8 bar to produce a resulting CO₂-expandedsolution, the reactor maintaining a pressure and temperature to preventthe CO₂ from going supercritical; and depressurizing the resultingCO₂-expanded solution over an aqueous phase to form vesicularconjugates, the resulting solution containing water soluble cationic ornon-ionic surfactants and hydrophilic or hydrophobic bio-actives,wherein an emulsion further comprises a water-soluble or fat-solublecosmetic or cosmeceutical active ingredient.
 2. The method of claim 1wherein the phospholipids are selected from the group consisting of: anonionic amphiphilic lipid, an ionic amphiphilic lipid, a mixture ofnonionic lipids, and an ionic lipid.
 3. The method of claim 1 whereinthe phospholipids are of the phosphate group of phosphatidylcholine (PC)amphiphilic molecules, having a polar head group and a lipophilic tail,and are esterified with an additional alcohol of choline possessing azwitterionic isoelectric point and are negatively charged at higher pHvalues and positively charged at lower pH values.
 4. The method of claim1 wherein the phospholipids are of the phosphate groupphosphatidylethanolarnine (PE), and are esterified with an additionalalcohol of ethanolarnine and are a neutral charged zwitterionic.
 5. Themethod of claim 1 wherein the phospholipids are of the phosphate groupphosphatidylglycerol (PG) with glycerol being anionic in charge.
 6. Themethod of claim 1 wherein the phospholipids are of the phosphate groupconsisting of a ceramide unit with a phosphorylcholine moiety attached,and a sphingolipid analogue of phosphatidylchol, or sphingomyelin, orceramide phosphorylcholine.
 7. The method of claim 1 wherein thephospholipids are amphiphilic liposomes wherein the molar ratio of themixture of cholesterol and at least one neutral or zwitterionic lipid isbetween 8 and 0.15.
 8. The method of claim 1 wherein the phospholipidsinclude at least one non-ionic amphiphilic lipid being an ester of atleast one polyol, said ester being a polyethylene glycol containing from0.5 to 70 ethylene oxide units, sorbitan, glycerol containing from 1 to35 ethylene oxide units, polyglycerol containing from 1 to 18 glycerolunits, or a fatty acid containing at least one saturated or unsaturated,linear or branched, C8-C22 alkyl chain.
 9. The method of claim 1 whereinat least one cationic amphiphilic lipid is present in the liposomesolution or mixture in a concentration ranging from 0.5 to 65% by weightwith respect to the total weight of the amphiphilic lipid phase.
 10. Themethod of claim 1 including using a formation of unilamellar vesicleshaving one lipid bilayer between 50-250 nm enclosing a large aqueouscore, and multilamellar vesicles having two or more concentric lipidbilayers between 1-5 μm.
 11. The method of claim 1 including usingcholesterol to anchor phosphate group phosphatidylcholine (PC)amphiphilic molecules, the phosphate group phosphatidylethanolarnine(PE) molecules, and the phosphate group phosphatidylglycerol (PG)molecules.
 12. The method of claim 1 including using an amphiphilicliposome having neutral lipids wherein said neutral lipids are selectedfrom the group consisting of cholesterol or mixtures of cholesterol andat least one neutral or zwitterionic lipid.
 13. The method of claim 1including using an amphiphilic liposome having phosphatidylcholineselected from DMPC, DPPC, DSPC, POPC, DOPC, soy bean PC or egg PC. 14.The method of claim 1 including using an amphiphilic liposome having Kneutral of a mixture of cholesterol and at least one neutral orzwitterionic lipids.
 15. The method of claim 1 including using anamphiphilic liposome having a mixture of cholesterol and at least oneneutral or zwitterionic lipid selected from the group consisting of:cholesterol/phosphatidylcholine, cholesterol/phosphatidylethanolamine,and cholesterol/phosphatidylethanolamine/ phosphatidylcholine.
 16. Themethod of claim 1 including using an amphiphilic liposome having amixture of lipid components with amphiphilic properties and wherein saidmixture of lipid components comprises at least one pH responsivecomponent.
 17. The method of claim 1 including using an amphiphilicliposome that encapsulates at least one active agent.
 18. The method ofclaim 1 including using an amphiphilic liposome wherein at least 80 wt.percentage of an active agent is disposed inside said liposomes.
 19. Themethod of claim 1 including liposomes being non-encapsulated activeagents.
 20. The method of claim 1 including forming a cosmeceuticalcomposition comprising active agent-loaded amphiphilic liposomes and acosmeceutically acceptable vehicle therefor.
 21. The method of claim 1including using amphiphilic liposomes in vitro, in vivo or ex-vivotransfection of cells.